WO2021100709A1 - 細胞構造体及びその製造方法並びに被験物質の肝毒性の評価方法 - Google Patents

細胞構造体及びその製造方法並びに被験物質の肝毒性の評価方法 Download PDF

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WO2021100709A1
WO2021100709A1 PCT/JP2020/042824 JP2020042824W WO2021100709A1 WO 2021100709 A1 WO2021100709 A1 WO 2021100709A1 JP 2020042824 W JP2020042824 W JP 2020042824W WO 2021100709 A1 WO2021100709 A1 WO 2021100709A1
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cell structure
cells
extracellular matrix
cell
matrix component
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PCT/JP2020/042824
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French (fr)
Japanese (ja)
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靖之 平岡
史朗 北野
新司 入江
典弥 松▲崎▼
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凸版印刷株式会社
国立大学法人大阪大学
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Priority to CN202080078503.4A priority Critical patent/CN114729307A/zh
Priority to EP20890313.8A priority patent/EP4063486A4/en
Priority to US17/777,786 priority patent/US20220411750A1/en
Priority to JP2021558396A priority patent/JPWO2021100709A1/ja
Publication of WO2021100709A1 publication Critical patent/WO2021100709A1/ja
Priority to JP2022130662A priority patent/JP2022163218A/ja

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • C—CHEMISTRY; METALLURGY
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to a cell structure, a method for producing the same, and a method for evaluating hepatotoxicity of a test substance.
  • DILI Drug-induced liver injury
  • a method for artificially producing a structure imitating a living tissue for example, a method for producing a three-dimensional tissue by culturing coated cells in which the entire surface of the cultured cells is covered with an adhesive film (Patent Document 1). ), The cells are mixed with a cationic substance and an extracellular matrix component to obtain a mixture, and the cells are collected from the obtained mixture to form a cell aggregate on a substrate, which comprises forming a cell aggregate on a substrate.
  • a manufacturing method (Patent Document 2) and the like are known.
  • the present inventors have proposed a method for producing a three-dimensional tissue having a high collagen concentration by contacting cells with endogenous collagen, preferably further with fibrous exogenous collagen (Patent Document 3). doing.
  • An object of the present invention is to provide a cell structure having excellent responsiveness to a substance having hepatotoxicity and a method for producing the same.
  • the present invention relates to, for example, the following inventions.
  • [1] Contains cells containing at least hepatocytes and vascular endothelial cells, and extracellular matrix components, The extracellular matrix component is arranged between the cells, A cell structure having a liver sinusoid network between the cells.
  • [2] The cell structure according to [1], wherein the ratio of the number of hepatocytes to the total number of the cells is 60% or more and 80% or less.
  • [3] The cell structure according to [1] or [2], wherein the ratio of the number of vascular endothelial cells to the total number of cells is 5% or more and 35% or less.
  • [4] The cell structure according to any one of [1] to [3], wherein the extracellular matrix component is fibrous.
  • [5] The cell structure according to any one of [1] to [4], wherein the extracellular matrix component contains a collagen component.
  • [6] The cell structure according to any one of [1] to [5], which comprises a polymer electrolyte.
  • [7] The cell structure according to any one of [1] to [6], wherein the extracellular matrix component contains a fragmented extracellular matrix component.
  • [8] The cell structure according to any one of [1] to [7], wherein the vascular endothelial cells are sinusoidal endothelial cells.
  • [9] The cell structure according to any one of [1] to [8], wherein the cell further contains hepatic stellate cells.
  • a culture step of culturing the cell structure according to any one of [1] to [9] in contact with a test substance is provided. Hepatotoxicity of the test substance is evaluated for the presence or absence or degree of hepatotoxicity using the cell survival number, albumin production amount, adenosine triphosphate content, or liver sinusoid network of the cell structure after the culture step as an index. Evaluation method.
  • a contact step in which cells containing at least hepatocytes and vascular endothelial cells and extracellular matrix components are brought into contact with each other in an aqueous medium.
  • a culture step of culturing the cells in contact with the extracellular matrix component is provided.
  • the contact step is performed under conditions in which aggregation of the extracellular matrix components in an aqueous medium is suppressed.
  • a method for producing a cell structure wherein the culture step is performed under conditions suitable for culturing hepatic nonparenchymal cells.
  • [14] The method for producing a cell structure according to [12] or [13], wherein the contact step is performed by mixing the cell, the extracellular matrix component, and a polymer electrolyte.
  • the contact step comprises accumulating the cell and the extracellular matrix component after the contact between the cell and the extracellular matrix component. Production method.
  • [18] The method for producing a cell structure according to any one of [12] to [17], wherein the extracellular matrix component contains a fragmented extracellular matrix component. [19] The method for producing a cell structure according to [18], wherein the fragmented extracellular matrix component is a defibrated extracellular matrix component. [20] The method for producing a cell structure according to any one of [12] to [19], wherein the vascular endothelial cells are sinusoidal endothelial cells. [21] The method for producing a cell structure according to any one of [12] to [20], wherein the cells further contain hepatic stellate cells. [22] The method for producing a cell structure according to any one of [12] to [21], wherein the culture step is performed in the presence of an angioplasty promoting factor.
  • the present invention it is possible to provide a cell structure having excellent responsiveness to a substance having hepatotoxicity and a method for producing the same.
  • the cell structure of the present invention can maintain liver function for a relatively long period of time (for example, 2 weeks or more). By using the cell structure of the present invention, it is possible to evaluate the hepatotoxicity of the test substance.
  • the cell structure according to the present embodiment contains a cell containing at least hepatocytes and vascular endothelial cells and an extracellular matrix, and the extracellular matrix is arranged between the cells.
  • the cell structure according to the present embodiment is a liver tissue (liver model) which is a biological tissue model having a function similar to the function of at least a part of the liver and / or a structure similar to the structure of at least a part of the liver. Can be used as.
  • the "cell structure” is an aggregate of cells in which cells are three-dimensionally arranged (massive cell population), and means an aggregate artificially produced by cell culture. Extracellular matrix components may be arranged between at least some cells. In the cell structure, there may be a portion where cells are in direct contact with each other.
  • the shape of the cell structure is not particularly limited, and is, for example, sheet-like, spherical, substantially spherical, ellipsoidal, approximately ellipsoidal, hemispherical, approximately hemispherical, semicircular, approximately semicircular, rectangular parallelepiped. , Approximately rectangular parallelepiped, etc.
  • the biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and is more complicated in composition than the cell structure. Therefore, the cell structure and the living tissue can be easily distinguished. Further, the cell structure may be assembled in a mass in a state of being adhered to the support, or may be aggregated in a mass in a state of not being adhered to the support.
  • the cells may be somatic cells or germ cells. Further, the cell may be a stem cell, or may be a cultured cell such as a primary cultured cell, a subcultured cell, and a cell line cell.
  • stem cell means a cell having self-renewal ability and pluripotency.
  • Stem cells include pluripotent stem cells capable of differentiating into arbitrary cell tumors and tissue stem cells (also called somatic stem cells) capable of differentiating into specific cell tumors.
  • pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells).
  • tissue stem cells include mesenchymal stem cells (eg, bone marrow-derived stem cells), hematopoietic stem cells, and neural stem cells.
  • the total number of cells constituting the cell structure according to the present embodiment is not particularly limited, and is appropriately considered in consideration of the thickness and shape of the cell structure to be constructed, the size of the cell culture vessel used for construction, and the like. It is determined.
  • the cells include at least hepatocytes and vascular endothelial cells.
  • Hepatocytes are also called hepatic parenchymal cells, and are cells having functions such as bile secretion and plasma protein secretion, for example.
  • the hepatocytes constituting the cell structure may be primary hepatocytes collected from the liver of an animal, cells obtained by culturing primary hepatocytes, or a cultured cell line obtained by culturing primary hepatocytes. It may be a hepatocyte artificially differentiated from a stem cell.
  • primary hepatocytes include primary human hepatocytes such as PXB Cell.
  • Examples of the cultured cell line include a cell line derived from inactivated liver cancer cells such as HepG2.
  • stem cells that differentiate into hepatic blast cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • mesenchymal stem cells mesenchymal stem cells.
  • non-cancerous cells such as primary hepatocytes and hepatoblasts are preferable, and PXB Cell is more preferable from the viewpoint of ease of handling.
  • the hepatocytes contained in the cell structure may be one type or two or more types.
  • the cell structure may contain a plurality of hepatocytes having different genotypes of proteins involved in liver function.
  • all hepatocytes contained in the cell structure may have the same genotype of the protein involved in liver function.
  • proteins involved in liver function include drug-metabolizing enzymes.
  • the ratio of the number of hepatocytes (X 1 ) to the total number of cells (X 0 ) in the cell structure (X 1 / X 0 ⁇ 100) is 5% or more, 10% or more, 15% or more, 20% or more, It may be 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, 95% or less, 90% or less, 80. It may be% or less or 75% or less.
  • the ratio (X 1 / X 0 ⁇ 100) of the number of hepatocytes (X 1 ) to the total number of cells (X 0 ) in the cell structure is 60% or more and 80 from the viewpoint of being more suitable as a liver tissue. % Or less, or 60% or more and 70% or less.
  • Endothelial cells mean flat cells that make up the surface of the vascular lumen.
  • the vascular endothelial cells may be, for example, sinusoidal endothelial cells, human umbilical vein-derived vascular endothelial cells (HUVEC).
  • Sinusoidal endothelial cells are non-hepatic parenchymal cells (cells other than hepatocytes among the cells constituting the liver), have a large number of small pore aggregates (screen plate structure) in the cytoplasm, lack the basal membrane, etc. It is a cell having a characteristic morphology different from that of vascular endothelial cells.
  • the vascular endothelial cells constituting the cell structure may be primary cells (primary vascular endothelial cells) collected from the liver of an animal (for example, human), cells obtained by culturing primary cells, or primary cells. It may be a cultured cell line in which cells are established, or may be cells artificially differentiated from stem cells. Examples of the primary vascular endothelial cells include primary sinusoidal endothelial cells such as product model number 5000 manufactured by Sciencell. Examples of the cultured cell line include a cultured cell line of product model number T0056 manufactured by Applied Biological Materials. Examples of stem cells to be differentiated include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The vascular endothelial cells contained in the cell structure according to the present embodiment may be non-cancerous cells.
  • primary vascular endothelial cells collected from the liver of an animal (for example, human), cells obtained by culturing primary cells, or primary cells. It may be a cultured cell line in
  • the ratio (X 2 / X 0 x 100) of the number of vascular endothelial cells (X 2 ) to the total number of cells (X 0 ) in the cell structure is 5% or more, 10% or more, 12% or more, 14% or more. , 15% or more, 20% or more, or 25% or more, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. May be.
  • the ratio (X 2 / X 0 ⁇ 100) of the number of vascular endothelial cells (X 2 ) to the total number of cells (X 0 ) in the cell structure is 5% or more from the viewpoint of being more suitable as a liver tissue.
  • the number of sinusoidal endothelial cells relative to the total number of cells in the cell structure may be within the above range.
  • the cells may further contain hepatic stellate cells from the viewpoint of being more suitable as a liver tissue.
  • Hepatocytes are non-hepatocytes (cells other than hepatocytes among the cells constituting the liver, have a function of storing vitamin A, etc., and are regions between hepatocytes and sinuses in the liver.
  • the hepatocytes present in the disse cavity may be, for example, primary cells (primary hepatocytes) collected from the liver of an animal (for example, human), or cells obtained by culturing primary cells. , A cultured cell line in which a primary cell is established, or a cell artificially differentiated from a stem cell.
  • the primary hepatocyte is, for example, a primary hepatocyte of model number 5300 manufactured by Sciencell.
  • Examples of the cultured cell line include a cultured cell line such as LX-2.
  • Examples of the stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and interstitial cells. Examples include hepatocytes.
  • the hepatocytes contained in the cell structure according to the present embodiment may be non-cancerous cells.
  • the ratio (X 3 / X 0 x 100) of the number of hepatic stellate cells (X 3 ) to the total number of cells (X 0 ) in the cell structure is 1% or more, 2% or more, 3% or more, 4% or more. , Or 5% or more, 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less.
  • the ratio (X 3 / X 0 ⁇ 100) of the number of hepatic stellate cells (X 3 ) to the total number of cells (X 0 ) in the cell structure is 1% or more from the viewpoint of being more suitable as a liver tissue. It may be 15% or less, or 3% or more and 12% or less.
  • the cells may include cells other than hepatocytes, vascular endothelial cells and hepatic stellate cells.
  • Other cells may be, for example, mature somatic cells or undifferentiated cells such as stem cells.
  • somatic cells include nerve cells, dendritic cells, immune cells, lymphatic endothelial cells, fibroblasts, epithelial cells (excluding hepatocytes), myocardial cells, pancreatic islet cells, and smooth muscle cells. , Bone cells, alveolar epithelial cells, spleen cells and the like.
  • stem cells include ES cells, iPS cells, mesenchymal stem cells and the like.
  • the other cell may be a normal cell, or may be a cell in which any cell function is enhanced or suppressed, such as a cancer cell.
  • a "cancer cell” is a cell derived from a somatic cell that has acquired infinite proliferative capacity.
  • the cells in the cell structure may or may not contain mesenchymal stem cells as other cells.
  • the ratio (X 4 / X 0 x 100) of the number of mesenchymal stem cells (X 4 ) to the total number of cells (X 0 ) in the cell structure is 5% or more, 10% or more, 15% or more, 20%. It may be 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, and may be 80% or less, 70% or less, 60% or less, or 55% or less.
  • hepatocytes vascular endothelial cells, hepatic stellate cells or other cells contained in the cells
  • mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice and rats. It may be a cell derived from an animal.
  • the cells in the cell structure according to the present embodiment do not have to contain cells induced to differentiate from induced pluripotent stem cells (iPS cells).
  • iPS cells induced pluripotent stem cells
  • blood vessels are formed between at least some cells.
  • “Blood vessels are formed between cells” means that the tubular structure formed by vascular endothelial cells has a structure extending between cells.
  • the cell structure may have gaps between cells in which no blood vessels are formed. The gap may be an empty space, but may be filled with an extracellular matrix component or the like.
  • the cell structure according to the present embodiment is expected to be able to be maintained for a long period of time because blood vessels are formed between cells like a living tissue. It is also expected that it will be easier to engraft when transplanted to mammals and the like.
  • the cell structure according to this embodiment preferably has a liver sinusoid network between cells.
  • "Having a hepatic sinusoid network” means that the tubular structure formed by vascular endothelial cells has a network-like structure formed so as to branch and surround the cells.
  • the vascular structure between cells and the liver sinusoid network can be confirmed by immunohistochemical staining.
  • the cell structure may have a liver sinusoid network to the extent that it has a plurality of bifurcation points and is observed to have a network structure when observed under a microscope from above.
  • the cell structure according to this embodiment contains an extracellular matrix component.
  • the extracellular matrix component is located between at least some cells.
  • the "extracellular matrix component” is an aggregate of extracellular matrix molecules formed by a plurality of extracellular matrix molecules.
  • the extracellular matrix means a substance that exists outside the cell in an organism.
  • any substance can be used as long as it does not adversely affect the growth of cells and the formation of cell aggregates.
  • Specific examples include, but are not limited to, collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, fibrillin, cadherin and the like.
  • the extracellular matrix component may be used alone or in combination of these.
  • the extracellular matrix component may contain, for example, a collagen component, and may be a collagen component.
  • the extracellular matrix component When the extracellular matrix component is a collagen component, the collagen component functions as a scaffold for cell adhesion, further promoting the formation of a three-dimensional cell structure.
  • the extracellular matrix component in the present embodiment is preferably a substance existing outside the animal cell, that is, an animal extracellular matrix component.
  • the extracellular matrix molecule may be a variant or variant of the above-mentioned extracellular matrix molecule as long as it does not adversely affect the growth of cells and the formation of cell aggregates, and is a polypeptide such as a chemically synthesized peptide. You may.
  • the extracellular matrix component may have a repeating sequence represented by Gly-XY, which is characteristic of collagen.
  • Gly represents a glycine residue
  • X and Y each independently represent an arbitrary amino acid residue.
  • the plurality of Gly-XY may be the same or different.
  • the proportion of the sequence represented by Gly-XY may be 80% or more, preferably 95% of the total amino acid sequence. That is all.
  • the extracellular matrix component may have an RGD sequence.
  • the RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue).
  • Arg-Gly-Asp arginine residue-glycine residue-aspartic acid residue.
  • the extracellular matrix component including the sequence represented by Gly-XY and the RGD sequence includes collagen, fibronectin, vitronectin, laminin, cadherin and the like.
  • the shape of the extracellular matrix component examples include fibrous form.
  • the fibrous form means a shape composed of a filamentous extracellular matrix component or a shape composed of a filamentous extracellular matrix component cross-linked between molecules. At least some of the extracellular matrix components may be fibrous.
  • the shape of the extracellular matrix component is the shape of a group of extracellular matrix components (aggregates of extracellular matrix components) observed when observed under a microscope, and the extracellular matrix components preferably have an average diameter and an average diameter described later. / Or has an average length.
  • the fibrous extracellular matrix component includes fine filaments (fine fibers) formed by aggregating a plurality of filamentous extracellular matrix molecules, filaments formed by further aggregating fine fibers, and these filaments. Deflated ones are included. When the extracellular matrix component having a fibrous shape is contained, the RGD sequence is preserved in the fibrous extracellular matrix component without being destroyed, and it functions more effectively as a scaffolding material for cell adhesion. be able to.
  • the extracellular matrix component may contain fragmented extracellular matrix components. "Fragmentation" means making the aggregate of extracellular matrix components smaller in size.
  • the fragmented extracellular matrix component may include a defibrated extracellular matrix component.
  • the defibrated extracellular matrix component is a component obtained by defibrating the above-mentioned extracellular matrix component by applying a physical force. For example, defibration is performed under conditions that do not break the bonds within the extracellular matrix molecule.
  • the fragmented extracellular matrix component can be produced, for example, by a method including a step of fragmenting the extracellular matrix component (fragmentation step).
  • the method for fragmenting the extracellular matrix component is not particularly limited, and may be fragmented by applying a physical force.
  • the extracellular matrix component fragmented by the application of physical force unlike enzymatic treatment, usually has the same molecular structure as before fragmentation (the molecular structure is maintained).
  • the method of fragmenting the extracellular matrix component may be, for example, a method of finely crushing the massive extracellular matrix component.
  • the extracellular matrix component may be fragmented in the solid phase or in an aqueous medium.
  • the extracellular matrix component may be fragmented by applying a physical force such as an ultrasonic homogenizer, a stirring homogenizer, and a high-pressure homogenizer.
  • the extracellular matrix component may be homogenized as it is, or it may be homogenized in an aqueous medium such as physiological saline. It is also possible to obtain fragmented extracellular matrix components of millimeter size and nanometer size by adjusting the homogenization time, the number of times, and the like.
  • the fragmented extracellular matrix component is, for example, a step of fragmenting the extracellular matrix component in an aqueous medium, and the fragmented extracellular matrix component and It can be produced by a method including a step of removing the aqueous medium from the liquid containing the aqueous medium (removal step).
  • the removal step may be carried out by, for example, a freeze-drying method.
  • "Removing the aqueous medium” does not mean that no water is attached to the fragmented extracellular matrix components, but can be reached by common sense by the above-mentioned general drying method. It means that there is not enough water to adhere to it.
  • the diameter and length of the fragmented extracellular matrix components can be determined by analyzing each fragmented extracellular matrix component with an electron microscope.
  • the average length of the fragmented extracellular matrix component may be 100 nm or more and 400 ⁇ m or less, and may be 100 nm or more and 200 ⁇ m or less. In one embodiment, the average length of the fragmented extracellular matrix components may be 5 ⁇ m or more and 400 ⁇ m or less, 10 ⁇ m or more and 400 ⁇ m or less, and 100 ⁇ m or more, from the viewpoint of facilitating the formation of thick cell structures. It may be 400 ⁇ m or less. In other embodiments, the average length of the fragmented extracellular matrix components may be 100 ⁇ m or less, 50 ⁇ m or less, 30 ⁇ m or less, 15 ⁇ m or less, and 10 ⁇ m or less.
  • the average length of most of the fragmented extracellular matrix components is within the above numerical range.
  • the average length of 50% or more of the fragmented extracellular matrix components is preferably within the above numerical range, and 95% of the fragmented extracellular matrix components are fragmented. It is more preferable that the average length of the extracellular matrix component is within the above numerical range.
  • the fragmented extracellular matrix component is preferably a fragmented collagen component having an average length within the above range.
  • the average diameter of the fragmented extracellular matrix components may be 50 nm to 30 ⁇ m, 4 ⁇ m to 30 ⁇ m, and 5 ⁇ m to 30 ⁇ m.
  • the fragmented extracellular matrix component is preferably a fragmented collagen component having an average diameter within the above range.
  • the average length and average diameter of the fragmented extracellular matrix components can be determined by measuring each fragmented extracellular matrix component with an optical microscope or the like and performing image analysis.
  • average length means the average value of the lengths of the measured samples in the longitudinal direction
  • average diameter means the average value of the lengths of the measured samples in the direction orthogonal to the longitudinal direction. means.
  • the fragmented collagen component is also called “fragmented collagen component”.
  • the "fragmented collagen component” means a fragmented collagen component such as a fibrous collagen component that maintains a triple helix structure.
  • the average length of the fragmented collagen component is preferably 100 nm to 200 ⁇ m, more preferably 22 ⁇ m to 200 ⁇ m, and even more preferably 100 ⁇ m to 200 ⁇ m.
  • the average diameter of the fragmented collagen component is preferably 50 nm to 30 ⁇ m, more preferably 4 ⁇ m to 30 ⁇ m, and even more preferably 20 ⁇ m to 30 ⁇ m.
  • the extracellular matrix component may be cross-linked between or within the molecule.
  • the extracellular matrix component may be cross-linked intramolecularly or between molecules of the extracellular matrix molecule constituting the extracellular matrix component.
  • the extracellular matrix component contains a fragmented extracellular matrix component
  • at least a part of the fragmented extracellular matrix component may be cross-linked intermolecularly or intramolecularly.
  • the extracellular matrix component which is at least partially cross-linked between or within the molecule, can be produced, for example, by a method including a step of cross-linking the extracellular matrix component (cross-linking step).
  • the extracellular matrix component can include, for example, fragmented and crosslinked extracellular matrix components.
  • the fragmented and cross-linked extracellular matrix component is, for example, a method comprising a step of fragmenting the extracellular matrix component and a step of cross-linking the fragmented extracellular matrix component in this order, or an extracellular matrix component.
  • cross-linking examples include physical cross-linking by applying heat, ultraviolet rays, radiation, etc., cross-linking agent, chemical cross-linking by enzymatic reaction, etc., but the method is not particularly limited. Physical cross-linking is preferable from the viewpoint of not hindering cell growth.
  • the cross-linking (physical cross-linking and chemical cross-linking) may be a cross-linking via a covalent bond.
  • the crosslinks may be formed between collagen molecules (triple helix structure) or between collagen fibrils formed by collagen molecules.
  • the cross-linking may be thermal cross-linking (thermal cross-linking). Thermal cross-linking can be carried out, for example, by performing heat treatment under reduced pressure using a vacuum pump.
  • thermally cross-linking a collagen component the extracellular matrix component is cross-linked by forming a peptide bond (-NH-CO-) with the carboxy group of the same or other collagen molecule in the amino group of the collagen molecule. It's okay.
  • the extracellular matrix component can also be crosslinked by using a crosslinking agent.
  • the cross-linking agent may be, for example, one capable of cross-linking a carboxyl group and an amino group, or one capable of cross-linking amino groups with each other.
  • As the cross-linking agent for example, aldehyde-based, carbodiimide-based, epoxide-based and imidazole-based cross-linking agents are preferable from the viewpoint of economy, safety and operability, and specifically, glutaraldehyde and 1-ethyl-3- (3).
  • Examples thereof include water-soluble carbodiimides such as -dimethylaminopropyl) carbodiimide / hydrochloride and 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide / sulfonate.
  • the degree of cross-linking can be appropriately selected according to the type of extracellular matrix component, the means for cross-linking, and the like.
  • the degree of cross-linking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and may be 30% or less, 20% or less, or 15% or less.
  • the degree of cross-linking is in the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after dry storage is good.
  • the degree of cross-linking is determined by Acta Biomateria, 2015, vol. 25, pp. It can be quantified based on the TNBS (2,4,6-trinitrobenzenesulfonic acid) method described in 131-142 and the like.
  • the degree of cross-linking by the TNBS method may be within the above range.
  • the degree of cross-linking by the TNBS method is the ratio of the amino groups used for cross-linking among the amino groups of the extracellular matrix.
  • the degree of cross-linking measured by the TNBS method is preferably within the above range.
  • the degree of cross-linking may be calculated by quantifying the carboxyl group. For example, in the case of an extracellular matrix component that is insoluble in water, it may be quantified by the TBO (toluidine blue O) method. The degree of cross-linking by the TBO method may be within the above range.
  • the temperature (heating temperature) and time (heating time) when heating the extracellular matrix component can be appropriately determined.
  • the heating temperature may be, for example, 100 ° C. or higher, 200 ° C. or lower, or 220 ° C. or lower.
  • the heating temperature is, for example, 100 ° C., 110 ° C., 120 ° C., 130 ° C., 140 ° C., 150 ° C., 160 ° C., 170 ° C., 180 ° C., 190 ° C., 200 ° C., 220 ° C., or the like. It's okay.
  • the heating time time to hold at the above heating temperature
  • the heating time may be, for example, 6 hours or more and 72 hours or less, more preferably 24 hours or more and 48 hours or less when heating at 100 ° C. to 200 ° C.
  • heating may be performed in the absence of a solvent, or heating may be performed under reduced pressure conditions.
  • the content of the extracellular matrix component in the cell structure is 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more based on the dry weight of the cell structure. 1, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass, It may be 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, It may be 30% by mass or less, 20% by mass or less, or 15% by mass or less.
  • the content of the extracellular matrix component in the cell structure may be 0.01 to 90% by mass, based on the dry weight of the cell structure, 10 to 90% by mass, 10 to 80% by mass, and 10 to 70. It may be% by mass, 10 to 60% by mass, 1 to 50% by mass, 10 to 50% by mass, 10 to 30% by mass, or 20 to 30% by mass.
  • extracellular matrix component in the cell structure means an extracellular matrix component constituting the cell structure, which may be derived from an endogenous extracellular matrix component, and may be derived from an endogenous extracellular matrix component. It may be derived from.
  • Intracellular extracellular matrix component means an extracellular matrix component produced by an extracellular matrix-producing cell.
  • extracellular matrix-producing cells include mesenchymal cells such as the above-mentioned fibroblasts, chondrocytes, and osteoblasts.
  • the endogenous extracellular matrix component may be fibrous or non-fibrous.
  • Extrinsic extracellular matrix component means an extracellular matrix component supplied from the outside.
  • the cell structure according to the present embodiment contains a fragmented extracellular matrix component which is an exogenous extracellular matrix component.
  • the exogenous extracellular matrix component may be the same as or different from the endogenous extracellular matrix component in the animal species from which it is derived. Examples of the animal species from which it is derived include humans, pigs, and cattle.
  • the exogenous extracellular matrix component may be an artificial extracellular matrix component.
  • the exogenous extracellular matrix component is also referred to as "exogenous collagen component", and there are a plurality of "exogenous collagen components” meaning collagen components supplied from the outside. It is an aggregate of collagen molecules formed by the collagen molecules of the above, and specific examples thereof include fibrous collagen and non-fibrous collagen.
  • the exogenous collagen component is preferably fibrous collagen.
  • the fibrous collagen means a collagen component which is a main component of collagen fibers, and examples thereof include type I collagen, type II collagen, and type III collagen.
  • commercially available collagen may be used, and specific examples thereof include porcine skin-derived type I collagen manufactured by Nippon Ham Co., Ltd.
  • exogenous non-fibrous collagen include type IV collagen.
  • the animal species from which it is derived may be different from the cell.
  • the animal species from which the extracellular matrix component is derived may be different from that of the extracellular matrix-producing cells. That is, the exogenous extracellular matrix component may be a heterologous extracellular matrix component.
  • the content of the extracellular matrix component constituting the cell structure is divided into the endogenous extracellular matrix component. It means the total amount of extracellular matrix components.
  • the extracellular matrix content can be calculated from the volume of the obtained cell structure and the mass of the decellularized cell structure.
  • the extracellular matrix component contained in the cell structure is a collagen component
  • the following method for quantifying hydroxyproline can be mentioned.
  • a sample is prepared by mixing hydrochloric acid (HCl) with the lysate in which the cell structure is dissolved, incubating at a high temperature for a predetermined time, returning to room temperature, and diluting the centrifuged supernatant to a predetermined concentration.
  • the hydroxyproline standard solution is treated in the same manner as the sample, and then diluted stepwise to prepare the standard.
  • Each of the sample and the standard is subjected to a predetermined treatment with a hydroxyproline assay buffer and a detection reagent, and the absorbance at 570 nm is measured.
  • the amount of collagen component is calculated by comparing the absorbance of the sample with the standard.
  • the cell structure may be directly suspended in high-concentration hydrochloric acid, and the dissolved solution may be centrifuged to recover the supernatant and used for quantifying the collagen component. Further, the cell structure to be lysed may be in a state of being recovered from the culture solution, or may be lysed in a state of being dried after the recovery to remove the liquid component.
  • the cells are affected by the medium component absorbed by the cell structure and the remaining influence of the medium due to the problem of the experimental procedure. Since the measured value of the structure weight is expected to vary, it is preferable to use the weight after drying as a reference from the viewpoint of stably measuring the weight of the tissue and the amount of collagen components per unit weight.
  • sample preparation The entire amount of the lyophilized cell structure is mixed with 6 mol / L HCl, incubated in a heat block at 95 ° C. for 20 hours or more, and then returned to room temperature. After centrifuging at 13000 g for 10 minutes, the supernatant of the sample solution is collected. A sample is prepared by appropriately diluting with 6 mol / L HCl so that the result falls within the range of the calibration curve in the measurement described later, and then diluting 200 ⁇ L with 100 ⁇ L of ultrapure water. 35 ⁇ L of sample is used.
  • the collagen component in the cell structure may be defined by its area ratio or volume ratio.
  • "Defined by area ratio or volume ratio” means that the collagen component in the cell structure is stained with another tissue by a known staining method (for example, immunostaining using an anti-collagen antibody or Masson's trichrome staining). It means that the ratio of the region where the collagen component is present in the entire cell structure is calculated by using macroscopic observation, various microscopes, image analysis software, etc. after making the cell distinguishable from the object.
  • the area ratio is not limited by what cross section or surface in the cell structure, but for example, when the cell structure is a spherical body or the like, a cross-sectional view passing through a substantially central portion thereof. May be specified by.
  • the ratio of the area is usually 0.01 to 99%, and 1 to 99%, based on the total area of the cell structure. It may be 5 to 90%, 7 to 90%, 20 to 90%, 30 to 90%, or 50 to 90%.
  • the "collagen component in the cell structure" is as described above.
  • the ratio of the area of the collagen component constituting the cell structure means the ratio of the total area of the endogenous collagen component and the extrinsic collagen component.
  • the ratio of the area of the collagen component is, for example, the ratio of the area of the collagen component stained blue to the total area of the cross section of the obtained cell structure stained with Masson Trichrome and passing through the substantially central part of the cell structure. It is possible to calculate.
  • the cell structure according to one embodiment may further contain a polymer electrolyte.
  • the polymer electrolyte is a polymer compound having the properties of an electrolyte.
  • examples of the polymer electrolyte include glycosaminoglycans such as heparan, chondroitin sulfate (for example, chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratane sulfate, and hyaluronic acid; Examples thereof include, but are not limited to, caraginan, polystyrene sulfonic acid, polyacrylamide-2-methylpropansulfonic acid, and polyacrylic acid, or derivatives thereof.
  • the polymer electrolyte may consist of one of the above-mentioned ones, or may contain a combination of two or more of the above-mentioned ones.
  • the polymer electrolyte is preferably glycosaminoglycan, more preferably containing at least one selected from the group consisting of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate, and further preferably heparin.
  • the ratio of the mass C 2 of the polymer electrolyte to the mass C 1 of the extracellular matrix component (C 2 / C 1 ) is 1/100 to 100/1, 1/10 to 10/1, 1/5 to 5 /. It may be 1 or 1/2 to 2/1, and may be 1 / 1.5 to 1.5 / 1.
  • the thickness of the cell structure may be 10 ⁇ m or more, 30 ⁇ m or more, 50 ⁇ m or more, 100 ⁇ m or more, 300 ⁇ m or more, or 1000 ⁇ m or more.
  • Such a cell structure has a structure closer to that of a living tissue, and is suitable as a substitute for an experimental animal and a material for transplantation.
  • the upper limit of the thickness of the cell structure is not particularly limited, but may be, for example, 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.
  • the thickness of the cell structure means the distance between both ends in the direction perpendicular to the main surface when the cell structure is in the form of a sheet or a rectangular parallelepiped.
  • the thickness means the distance at the thinnest part of the main surface.
  • the thickness of the cell structure means the diameter of the cell structure.
  • the thickness of the cell structure means the minor axis of the cell structure.
  • the thickness of the cell structure is the distance between the two points where the straight line passing through the center of gravity of the cell structure and the surface intersect. It means the shortest distance.
  • the cell structure preferably contains a fragmented extracellular matrix component and / or contains a polymeric electrolyte. In this case, the cell structure becomes even more suitable as a tissue model for evaluating hepatotoxicity.
  • Fibrin The cell structure according to this embodiment may contain fibrin.
  • Fibrin is a component produced by the action of thrombin on fibrinogen to release the A and B chains from the N-terminus of the A ⁇ and B ⁇ chains.
  • Fibrin is a polymer and is generally insoluble in water. Fibrin is formed by contacting fibrinogen with thrombin.
  • the cell structure according to this embodiment is constructed in a cell culture vessel.
  • the cell culture container is not particularly limited as long as it is a container capable of constructing a cell structure and capable of culturing the constructed cell structure.
  • Specific examples of the cell culture vessel include dishes, cell culture inserts (eg, Transwell® insert, Netwell® insert, Falcon® cell culture insert, Millicell® cell culture. Inserts, etc.), tubes, flasks, bottles, plates, etc.
  • a dish or various cell culture inserts are preferable from the viewpoint that the evaluation using the cell structure can be performed more appropriately.
  • the method for producing a cell structure according to the present embodiment is a contact step in which a cell containing at least hepatocytes and vascular endothelial cells and an extracellular matrix component are brought into contact with each other in an aqueous medium, and the extracellular matrix component is brought into contact with the cell. It comprises a culturing step of culturing the cells.
  • the contact step may be performed under conditions in which aggregation of extracellular matrix components in an aqueous medium is suppressed, and the culture step is a culture of hepatic nonparenchymal cells. It may be carried out under the conditions suitable for.
  • contacting process In the contacting step, cells containing at least hepatocytes and vascular endothelial cells and extracellular matrix components are brought into contact with each other in an aqueous medium. By contacting the cell with the extracellular matrix component, it is expected that a three-dimensional cell structure can be easily obtained.
  • the contact between the cell and the extracellular matrix component may be performed, for example, under the condition that the aggregation of the extracellular matrix component in the aqueous medium is suppressed.
  • the liver sinusoid network is more likely to be formed in the cell structure.
  • the cells when the cells contain hepatocytes, the hepatocytes easily adhere to each other, so that the gap between the cells tends to be small, and as a result, it becomes difficult to form a tube-like structure like a liver sinusoid network. Conceivable.
  • the extracellular matrix component is, for example, present with the extracellular matrix component and a component that suppresses aggregation of the extracellular matrix component (for example, the above-mentioned polymer electrolyte), and / or at least one of the extracellular matrix components.
  • Aggregation in an aqueous medium can be suppressed by using a fragmented extracellular matrix component or the like as a portion.
  • the conditions under which the aggregation of the extracellular matrix component in the aqueous medium is suppressed are, for example, a condition in which a polymer electrolyte is present and / or a condition in which the extracellular matrix component is fragmented and contains an extracellular matrix. Good.
  • Aqueous medium means a liquid containing water as an essential component.
  • the aqueous medium may be, for example, an aqueous medium containing a cationic substance.
  • the aqueous medium containing the cationic substance is, for example, Tris-hydrochloride buffer, Tris-maleic acid buffer, Bis-Tris-buffer, or HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid).
  • a cationic buffer solution such as, and is a medium containing a cationic compound such as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, polyarginine and water as a cationic substance. You may.
  • a medium can also be used as the aqueous medium. Examples of the medium include liquid media such as Dulbecco's Modified Eagle's medium (DMEM) and vascular endothelial cell dedicated medium (EGM2).
  • DMEM Dulbecco's Modified Eagle's medium
  • ECM2 vascular endothelial cell dedicated medium
  • the liquid medium may be a mixed medium in which two types of media are mixed.
  • the concentration and pH of the cationic substance (for example, Tris in Tris-hydrochloric acid buffer) in an aqueous medium containing the cationic substance is not particularly limited as long as it does not adversely affect the growth of cells and the construction of cell structures.
  • the concentration of the cationic substance may be 10 to 100 mM, 40 to 70 mM, or 50 mM based on the total amount of the aqueous medium containing the cationic substance.
  • the pH of the aqueous medium eg, cationic buffer
  • a method of adding an aqueous medium containing an extracellular matrix component to a liquid, a method of adding cells to an aqueous medium containing an extracellular matrix component, a method of adding an extracellular matrix component and cells to an aqueous medium prepared in advance, and the like. can be mentioned.
  • the order in which the cells are brought into contact with the extracellular matrix component is not particularly limited. For example, after contacting some cells with the extracellular matrix component, the remaining cells are brought into contact with the extracellular matrix component. Alternatively, all cells may be contacted with extracellular matrix components simultaneously or substantially simultaneously.
  • the aqueous medium containing cells and extracellular matrix may or may not be mixed by stirring or the like after the addition of each substance.
  • the contacting step may include contacting the cells and extracellular matrix components and then incubating for a period of time.
  • the contact step may be performed after the cells have been accumulated in an aqueous medium. That is, the contact step may be performed by accumulating cells in an aqueous medium and then contacting the extracellular matrix components. By contacting the accumulated cells with the extracellular matrix component, it becomes easy to prepare a cell structure having a high cell density in the lower layer.
  • the cells can be accumulated by, for example, a method such as centrifugation or natural sedimentation.
  • the ratio of the number of hepatocytes (X 1 / X 0 ⁇ 100) to the total number of cells (X 0 ) is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30 % Or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, 95% or less, 90% or less, 80% or less or 75% It may be:
  • the ratio of the number of hepatocytes to the total number of cells (X 0) (X 1) (X 1 / X 0 ⁇ 100) from the viewpoint becomes more suitable than as an off-liver tissue, 60% 80% It may be 60% or more and 70% or less.
  • the ratio of the number of vascular endothelial cells (X 2 ) to the total number of cells (X 0 ) (X 2 / X 0 ⁇ 100) is 5% or more, 10% or more, 12% or more, 14% or more. , 15% or more, 20% or more, or 25% or more, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. May be.
  • the ratio (X 2 / X 0 ⁇ 100) of the number of vascular endothelial cells (X 2 ) to the total number of cells (X 0 ) in the cell structure is 5% or more from the viewpoint of being more suitable as a liver tissue. It may be 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, 10% or more and 25% or less, or 12% or more and 20% or less.
  • the ratio of the number of hepatic stellate cells (X 3 ) to the total number of cells (X 0 ) (X 3 / X 0 ⁇ 100) is 1% or more and 2% in the contact step. It may be 3% or more, 4% or more, or 5% or more, and may be 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less.
  • the ratio (X 3 / X 0 ⁇ 100) of the number of hepatic stellate cells (X 3 ) to the total number of cells (X 0 ) in the cell structure is 1% or more from the viewpoint of being more suitable as a liver tissue. It may be 15% or less, or 3% or more and 12% or less.
  • ratio (X 5 / X 0 ⁇ 100 ) of the total number of cells (X 0), hepatocytes, the number of other cells other than vascular endothelial cells and hepatic stellate cells (X 5) is more than 5% It may be 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, 80% or less, 70% or less, 60% or less, Or it may be 55% or less.
  • the ratio of the number of mesenchymal stem cells to the total number of cells (X 4) may be within the above range.
  • the above cells are not derived from iPS cells.
  • cells that are not derived from iPS cells are used, it becomes easier to identify the type and content of cells in the cell structure, and as a result, it is more suitable as a tissue model for evaluating hepatotoxicity. Become.
  • the concentration of the extracellular matrix component in the contact step can be appropriately determined according to the shape and thickness of the target cell structure, the size of the incubator, and the like.
  • the concentration of the extracellular matrix component in the aqueous medium in the contact step may be 0.1 to 90% by mass or 1 to 30% by mass.
  • the amount of extracellular matrix component in the contact step is, for example, 0.1 to 100 mg, 0.5 to 50 mg, 0.8 to 25 mg, 1.0 to 10 mg, for 1.0 ⁇ 10 6 cells cells. It may be 1.0 to 5.0 mg, 1.0 to 2.0 mg, or 1.0 to 1.8 mg, and may be 0.7 mg or more, 1.1 mg or more, 1.2 mg or more, 1.3 mg or more, or 1 It may be 4.0 mg or less, 3.0 mg or less, 2.3 mg or less, 1.8 mg or less, 1.7 mg or less, 1.6 mg or less, or 1.5 mg or less. The amount of fragmented extracellular matrix components in the contact step may be within the above range.
  • the mass ratio of extracellular matrix component to cell is preferably 1/1 to 1000/1, more preferably 9/1 to 900/1. It is more preferably 10/1 to 500/1.
  • the contact step may be, for example, a step of mixing the cells, the extracellular matrix component, and the polymer electrolyte in an aqueous medium (contact step A).
  • the concentration of the polymer electrolyte is more than 0 mg, 0.001 mg or more, 0.005 mg or more, 0.01 mg or more, 0.025 mg or more, 0.05 mg or more, or 0.075 mg with respect to the total amount of 1 mL of the aqueous medium. It may be more than 1.0 mg, 0.5 mg or less, or 0.1 mg or less.
  • the first contact step includes, for example, an extracellular matrix component-containing solution containing an extracellular matrix component and a first aqueous medium, a polymer electrolyte-containing solution containing a polymer electrolyte and a second aqueous medium, and cells. May be done by mixing.
  • the extracellular matrix component-containing solution the extracellular matrix component may be dissolved or dispersed in the first aqueous medium.
  • the polymer electrolyte-containing liquid the polymer electrolyte may be dissolved in a second aqueous medium.
  • the first aqueous medium and the second aqueous medium may be the same type of aqueous medium or different types of aqueous medium.
  • the order in which the extracellular matrix component-containing liquid, the polymer electrolyte-containing liquid, and the cells are mixed is not particularly limited, and may be mixed in any order.
  • a mixed solution in which an extracellular matrix component-containing solution and a polymer electrolyte-containing solution are mixed in advance may be prepared, and the mixed solution may be mixed with cells. Also, all may be mixed substantially at the same time.
  • the content of the extracellular matrix component is 0.001 to 1.5 mg / mL, 0.05 to 1.5 mg / mL, or 0 with respect to the total amount of the extracellular matrix component-containing solution. It may be 1 to 1.0 mg / mL.
  • the content of the polymer electrolyte is 0.001 to 10.0 mg / mL, 0.05 to 5.0 mg / mL, or 0.1 with respect to the total amount of the polymer electrolyte-containing liquid. It may be ⁇ 1.0 mg / mL.
  • the ratio (A 1 : A 2 ) of the mass A 1 of the polymer electrolyte to the mass A 2 of the extracellular matrix component in the contact step may be 1: 200 to 200: 1, and may be 1: 100 to 100 :. It may be 1, it may be 1:10 to 10: 1, it may be 1: 5 to 5: 1, it may be 1: 2 to 2: 1, and it may be 1: 1.5 to 1. It may be 5: 1 or 1: 1.
  • the contact step may be a step of bringing the fragmented extracellular matrix component into contact with the cell (contact step B).
  • the fragmented extracellular matrix component those described above can be used.
  • the second contact step may be performed by using the extracellular matrix component fragmented in part or all of the extracellular matrix component.
  • Fibrinogen and / or thrombin may be included in the contact step, or after the contact step and before the culture step. Fibrinogen and thrombin may be added at the same time, for example, one of them may be added first, and then the other may be added.
  • a first solution containing extracellular matrix components an aqueous medium and fibrinogen may be mixed with a second solution containing cells, an aqueous medium and thrombin.
  • the contact step may include accumulating cells and extracellular matrix components after contact between the cells and the extracellular matrix components. By accumulating these components, the distribution of extracellular matrix components and cells in the cell structure becomes more uniform. Examples of the method for accumulating cells and extracellular matrix components include a method of centrifuging a culture solution containing extracellular matrix components and cells, and a method of spontaneously precipitating.
  • Culture process In the culturing step, cells in contact with extracellular matrix components are cultivated. Culturing of cells in contact with the extracellular matrix is performed under conditions suitable for culturing hepatic nonparenchymal cells. Conditions suitable for culturing hepatic nonparenchymal cells mean conditions in which cells other than hepatocytes (for example, sinusoidal endothelial cells) are more likely to grow than hepatocytes.
  • the culturing step may be performed by culturing the above cells in, for example, a medium containing a medium for non-parenchymal cells of the liver, and in a medium containing no medium for non-parenchymal cells and containing a medium for non-parenchymal cells. Then, it may be carried out by culturing the above cells.
  • the medium used in the culture step does not have to contain insulin and transferrin, which are proteins secreted from the liver.
  • the medium used in the culture step include a medium for vascular endothelium (for example, EGM2 (manufactured by Lonza), EGM2-MV (manufactured by Lonza), Endothelial Cell Growth Medium 2 (manufactured by Promocell), Endothelial Cell Growth Medium. (Manufactured by Promocell), ECM (manufactured by Sciencell), and the medium may be a medium to which serum is added or a serum-free medium.
  • the medium is a mixture of two types of media. It may be a mixed medium. For example, it may be a mixed medium in which a medium for vascular endothelium and a medium for proliferating mesenchymal stem cells are mixed.
  • the culture step may be performed in the presence of an angioplasty promoting factor.
  • an angioplasty promoting factor As a medium for culturing the above cells, a medium containing an angiogenesis-promoting factor may be used.
  • angiogenesis-promoting factors include vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).
  • the culture temperature in the culture step may be, for example, 20 ° C to 40 ° C or 30 ° C to 37 ° C.
  • the pH of the medium may be 6-8 or 7.2-7.4.
  • the culturing time may be 1 day to 2 weeks, or 1 week to 2 weeks.
  • the incubator (support) is not particularly limited, and may be, for example, a plate having a bottom shape such as a dish, a well insert, a low adhesive plate, a U shape, or a V shape.
  • the cells may be cultured while being adhered to the support, the cells may be cultured without being adhered to the support, or the cells may be separated from the support and cultured in the middle of the culture.
  • a U-shaped, V-shaped, or other bottom surface shape that inhibits the adhesion of the cells to the support is formed. It is preferable to use a plate having or a low adsorption plate.
  • the cell density in the medium in the culture step can be appropriately determined according to the shape and thickness of the target cell structure, the size of the incubator, and the like.
  • the cell density in the medium in the culture step may be 1 to 10 8 cells / mL and may be 10 3 to 10 7 cells / mL.
  • the cell density in the medium in the culture step may be the same as the cell density in the aqueous medium in the contact step.
  • first culturing step After the above-mentioned culturing step (hereinafter, also referred to as “first culturing step”; the first contacting step is also referred to as “first contacting step”), a step of further contacting cells (second contacting step). , The step of culturing the cells (second culturing step) may be included.
  • the cells in the second contact step and the second culture step may be the same species as the cells used in the first contact step and the first culture step, or may be heterologous.
  • a cell structure having a two-layer structure can be prepared. Further, by repeatedly including the contact step and the culture step, a multi-layered cell structure can be prepared, and a more complicated tissue close to a living body can be prepared.
  • a cell structure in which blood vessels are formed between cells and a cell structure having a liver sinusoid network between cells can be preferably produced.
  • the cell structure according to the present embodiment can be applied as a substitute for an experimental animal, a transplant material, or the like, and specific examples thereof include tissue reconstruction, pathological in vitro model, and drug screening (drug evaluation). , Can be applied to assay screening of cosmetics and the like.
  • the cell structure according to the present embodiment Since the cell structure according to the present embodiment has excellent responsiveness to a substance having hepatotoxicity, it can be suitably used as a tool for evaluating the presence or absence or degree of hepatotoxicity of the test substance. By using the cell structure according to the present embodiment, it becomes easy to obtain a more reliable evaluation of the hepatotoxicity of the test substance. By using the method for evaluating hepatotoxicity of a test substance using the cell structure according to the present embodiment, it is possible to screen for a substance having hepatotoxicity.
  • the method for evaluating hepatotoxicity of a test substance includes a culturing step of culturing the above-mentioned cell structure in contact with the test substance.
  • the method for evaluating hepatotoxicity is not particularly limited, and for example, other assay methods (MTT assay, MTS assay) that can obtain values that correlate with the cell viability, ATP content, albumin production, and viable cell number of the cell structure. , Etc.), methods such as quantification of bile acid uptake by hepatocytes, quantification of glutathione, or markers used in clinical evaluation of the presence or absence of liver damage such as ALT and bilirubin. It can be appropriately selected according to the order.
  • the number of cells alive, albumin production, or adenosine triphosphate (ATP) content of the cell structure after the culture step is used as an index for hepatotoxicity. You may evaluate the presence or absence or degree of.
  • the presence or absence or degree of hepatotoxicity may be evaluated using the liver sinusoid network (vascular network) of the cell structure after the culture step as an index.
  • the hepatic sinusoid network is used as an index, specifically, for example, the degree of fragmentation of the hepatic sinusoid network and / or the total blood vessel length of the hepatic sinusoid network may be used as an index.
  • the contact between the cell structure and the test substance can be performed, for example, by adding the test substance to the culture medium of the cell structure.
  • the test substance may be, for example, a drug suspected of having hepatotoxicity.
  • the test substance to be evaluated may be one type or two or more types.
  • each compound may be evaluated by being in contact with a cell structure, or a plurality of compounds may be evaluated by being in contact with a cell structure at the same time.
  • the culturing step may be carried out by culturing the cell structure in a medium containing the test substance.
  • the time for culturing the cell structure in the medium containing the test substance is not particularly limited, and may be, for example, 24-96 hours, 48-96 hours, or 48-72 hours. If necessary, hydrodynamic addition such as reflux can be given as long as the culture environment is not significantly changed.
  • the method for evaluating the presence or absence or degree of hepatotoxicity using the number of surviving cells of the cell structure as an index after the culturing step can be performed by, for example, the following method.
  • the test substance When the number of viable hepatocytes in the cell structure is small (low viability) as compared to the case where the cell structure is cultured in the absence of the test substance, the test substance is transferred to the cell structure. It is evaluated to be toxic to the hepatocytes contained, that is, hepatotoxic. It can be evaluated that the greater the decrease in the survival rate of hepatocytes, the stronger the hepatotoxicity as compared with the case in the absence of the test substance. On the other hand, when the number of viable hepatocytes is about the same or significantly higher (survival rate is about the same or higher) as compared with the case of culturing in the absence of the test substance, the test substance is hepatotoxic. Evaluate that there is no.
  • the number of viable hepatocytes can be evaluated using a signal that correlates with the viable cells of hepatocytes or their abundance. It suffices if the number of viable hepatocytes at the time of evaluation can be measured, and it is not always necessary to measure the number in a living state.
  • hepatocytes can be labeled to distinguish them from other cells, and the signal from the label can be used as an index for examination. For example, by fluorescently labeling hepatocytes and then determining whether the cells are alive or dead, the living hepatocytes in the cell structure can be directly counted. At this time, an image analysis technique can also be used.
  • the cell life / death determination can be performed by a known cell life / death determination method such as trypan blue staining or PI (Propidium Iodide) staining.
  • the fluorescent label of hepatocytes is, for example, immunostaining using a fluorescently labeled secondary antibody that specifically binds to the primary antibody, with an antibody against a substance specifically expressed on the cell surface of the hepatocyte as the primary antibody. It can be performed by a known method such as a method.
  • the cell life / death determination and the measurement of the number of viable cells may be performed in the state of the cell structure or in the state where the cell structure is destroyed at the single cell level.
  • FACS fluorescence activated cell sorting
  • the number of viable hepatocytes in the cell structure can be measured by labeling the hepatocytes in the cell structure in a living state and detecting the signal from the label over time. It can.
  • the hepatocytes in the cell structure may be labeled after the cell structure is constructed, or the hepatocytes may be labeled in advance before the cell structure is constructed.
  • the fluorescence intensity of lysate obtained by lysing the cell structure can also be measured with a microplate reader or the like to obtain hepatocytes.
  • the number of living cells can be evaluated.
  • the method for evaluating the presence or absence or degree of hepatotoxicity using the albumin production amount of the cell structure as an index after the culturing step can be performed by, for example, the following method.
  • the test substance When the amount of albumin produced in the cell structure is low (albumin-producing ability is low) or the ATP content in the cell structure is low as compared with the case of culturing in the absence of the test substance, the test substance Is toxic to hepatocytes contained in the cell structure at the concentration, that is, is evaluated as hepatotoxic. It can be evaluated that the greater the decrease in albumin production or the decrease in ATP content, the stronger the hepatotoxicity, as compared with the case in the absence of the test substance. On the other hand, when the albumin production amount is the same or significantly higher (albumin production ability is the same or higher) or the ATP content is the same or significantly higher than when cultured in the absence of the test substance. The test substance is evaluated to be non-hepatotoxic at that concentration.
  • the amount of albumin produced can be evaluated, for example, by measuring albumin in the culture supernatant using an ELISA method.
  • Hepatocytes contained in the cell structure preferably have a common genotype of at least one type of drug-metabolizing enzyme, and more preferably have a common genotype of all drug-metabolizing enzymes. Since hepatotoxicity tends to depend on drug-metabolizing enzymes, hepatotoxicity can be evaluated more accurately by using a cell structure containing hepatocytes having a homozygous genotype of the drug-metabolizing enzyme. ..
  • a method for evaluating the presence or absence or degree of hepatotoxicity using the liver sinusoid network of the cell structure as an index after the culture step is, for example, quantifying the total blood vessel length of the liver sinusoid network in the cell structure after the culture step. It can be carried out by a method including the step of culturing. As a method for quantifying the total blood vessel length of the liver sinusoid network, for example, the method described in Examples described later can be used.
  • the method for evaluating the hepatotoxicity of the test substance according to the present embodiment can evaluate the hepatotoxicity using the liver sinusoid network in the cell structure as an index, and therefore, the hepatotoxicity of the test substance can be evaluated with even higher sensitivity. It is possible to evaluate the presence and degree.
  • the method for evaluating hepatotoxicity of a test substance according to the present embodiment is particularly effective in evaluating a test substance that is presumed to affect the liver sinusoid network.
  • ⁇ Test Example 1 Preparation of cell structure 1> Heparin (manufactured by Sigma) was dissolved in 20 mM Tris-hydrochloric acid buffer (pH 7.4) to obtain a heparin solution. Based on the total mass of the heparin solution, the heparin content was 1.0 mg / mL. Collagen (collagen type I, manufactured by Nippi) was dissolved in a 5 mM acetic acid solution to obtain a collagen solution. Based on the total mass of the collagen solution, the collagen content was 0.4 mg / mL.
  • the cell numbers of the various cells were adjusted to the ratios shown in Table 1.
  • the total number of cells per well was about 100,000 cells.
  • the obtained suspension was centrifuged at 25 ° C. and 400 ⁇ g for 1 minute. As a result, a viscous body in which collagen and heparin were attached to the cell surface was formed. After stretching, the supernatant was removed, and 250 ⁇ L of a mixed medium (mass ratio 1: 1) of vascular endothelial medium 1 (ECM) and mesenchymal stem cell growth medium (MSCGM2) was added into the microtube. The resulting suspension was seeded in 96-well cell culture inserts (Roche Inc, E-Plate Insert 16). After sowing , the cells were cultured in a CO 2 incubator (37 ° C., 5% CO 2 ) for a predetermined period to obtain a cell structure.
  • ECM vascular endothelial medium 1
  • MSCGM2 mesenchymal stem cell growth medium
  • FIGS. 1 to 3 are photographs showing the observation results of the cell structures of Examples 1 to 3.
  • FIG. 1 shows the observation results of the sample of Example 1 immunostained with CD31 and albumin after being fixed on the 7th day from the start date of culture.
  • FIG. 2 shows the observation results of the sample of Example 2 immunostained with CD31 and albumin after being fixed on the 8th day from the culture start date.
  • FIG. 3 shows the observation results of a sample of an example that was immunostained with CD31 and albumin after being fixed on the 8th day from the start date of culture.
  • the cell structures shown in FIGS. 1 to 3 had a liver sinusoid network.
  • ⁇ Test Example 2 Preparation of cell structure 2> [Preparation of defibrated collagen component]
  • a collagen component crosslinked collagen component in which at least a part of the collagen was crosslinked was obtained. No significant change in appearance was observed in collagen before and after heating at 200 ° C.
  • 50 mg of the crosslinked collagen component was placed in a 15 mL tube, 5 mL of ultrapure was added, and homogenized with a homogenizer (As One Corporation VH-10) for 6 minutes.
  • the aqueous solution containing the homogenized crosslinked collagen component was centrifuged at 10000 rpm for 10 minutes under the condition of 21 ° C. The supernatant was aspirated and the collagen pellet was mixed with 5 mL of fresh ultrapure water to make a collagen solution. While keeping the tube containing the collagen solution on ice, ultrasonically treat it at 100 V for 20 seconds using a sonicator (Sonics and Materials VC50), and after removing the sonicator, cool the tube containing the collagen solution on ice for 10 seconds. This was repeated 100 times.
  • a sonicator Sonics and Materials VC50
  • the collagen solution was filtered through a filter having a pore size of 40 ⁇ m to obtain a dispersion containing the defibrated collagen component (sCMF).
  • the dispersion was freeze-dried by a conventional method to obtain a defibrated collagen component (sCMF) as a dried product.
  • a dispersion A was obtained in which the defibrated collagen component was dispersed so that the concentration was 30 mg / mL in a medium (DMEM) containing serum in which 10 mg / mL fibrinogen (manufactured by Sigma) was dissolved. Further, in a medium (DMEM) containing serum in which 10 U / mL thrombin (manufactured by Sigma) was dissolved, a dispersion B in which cells were dispersed so that the total number of cells per well was 30,000 cells was obtained. It was.
  • a suspension in which the obtained dispersion liquid A (defibrated collagen component) and the dispersion liquid B were mixed at a ratio of 1: 1 was added to a 48-well plate in an amount of 10 ⁇ L per well.
  • the observation results of the culture containing the cells and the defibrated collagen component after 6 days from the start of the culture are shown in FIGS. 4 to 6.
  • the medium is a mixed medium (mass ratio: 1: 1) of a medium for vascular endothelium 1 (ECM) and a medium for mesenchymal stem cell proliferation (MSCGM2), a medium for vascular endothelium 1 (ECM), or a medium for vascular endothelium 1. (ECM) and hepatocellular medium (d-HCGM) were used.
  • ⁇ Test Example 3 Preparation of cell structure 3> A mixture of 100 ⁇ L of heparin solution and 100 ⁇ L of collagen solution, PXB cell, SEC and Lx2, and MSC, if necessary, were placed in a microtube and suspended. The cell numbers of the various cells were adjusted to the ratios shown in Table 3. The total number of cells was about 30,000 cells.
  • the obtained suspension was centrifuged at 25 ° C. and 400 ⁇ g for 1 minute. As a result, a viscous body in which collagen and heparin were attached to the cell surface was formed. After stretching, the supernatant was removed, and 250 ⁇ L of a mixed medium (mass ratio 1: 1) of vascular endothelial medium 1 (ECM) and mesenchymal stem cell growth medium (MSCGM2) was added into the microtube. The resulting suspension was seeded in 96-well cell culture inserts (Roche Inc, E-Plate Insert 16). After sowing , the cells were cultured in a CO 2 incubator (37 ° C., 5% CO 2 ) for a predetermined period to obtain a cell structure.
  • ECM vascular endothelial medium 1
  • MSCGM2 mesenchymal stem cell growth medium
  • ⁇ Test Example 4 Preparation of cell structure 4>
  • the cell structure of Example 8 was prepared in the same manner as in Condition 1 of Test Example 1 except that the medium was changed to the medium for vascular endothelium 2 (EGM2MV) and the medium was fixed on the 7th day of the culture period. The results are shown in FIG. It was confirmed that the cell structures obtained using different media also formed a liver sinusoid network.
  • EMM2MV vascular endothelium 2
  • Example 5 Measurement of albumin secretion>
  • the amount of albumin secreted was measured by quantifying the amount of albumin in the culture supernatant during the culture period by ELISA. The medium was exchanged on the day before the supernatant collection day, and the culture supernatant for 24 hours was used as a sample for ELISA measurement.
  • a negative control a cell structure prepared in the same manner as in Example 1 was prepared except that it did not contain PXB Cell.
  • the measurement result of the albumin secretion amount is shown in FIG. At 15 days after the start of culture, significant maintenance of albumin secretion was confirmed with respect to negative control.
  • Example 6 Evaluation of hepatotoxicity> The hepatotoxicity evaluation test was prepared by the methods of Example 5 and Comparative Example 1, and the cell structure 7 days after the start of culturing was used as the evaluation cell structure. For the evaluation of hepatotoxicity, Nefazodone and Troglitazone, which are known to have hepatotoxicity, were used.
  • the cell structure having the liver sinusoid network is superior in responsiveness to substances having hepatotoxicity as compared with the cell structure without the liver sinusoid network.
  • the cells were collected according to the following procedure and the cell mass was measured.
  • the PXBcell was washed with PBS. 0.25% trypsin-EDTA 2 mL was added to PXBcell and incubated in an incubator. Then, HCGM was added to the PXB cell, pipetting was performed, the PXB cell was collected, and the measurement was performed with a cell counter.
  • PXB cell, SEC and LX2 were mixed so as to have the following ratio and cell mass to obtain a cell mixture.
  • -Total cell volume per well 30,000 cells ⁇
  • Tissue cell ratio PXB cells: 65%, SEC: 25%, LX2: 10%
  • heparin-collagen solution An equal amount of a 1.0 mg / mL heparin solution (buffer: 100 mM Tris-HCL) and a 0.3 mg / mL collagen solution (buffer: 5 mM acetate) were mixed to prepare a heparin-collagen solution.
  • heparin / collagen solution 100 ⁇ L was added to the cell mixture, suspended until the cells became invisible, and then centrifuged (400 g ⁇ 2 min) to form a viscous body in the solution.
  • a 20 U / mL trombine solution (solvent: HCM (for hepatocytes) so that the final volume of the solution is the "planned number of wells to be seeded" x 2 ⁇ L. Culture medium)) was added to obtain a cell suspension.
  • a 10 mg / mL fibrinogen solution was placed on 48 plates to form droplets.
  • a cell suspension was added to the inside of the droplet and then allowed to stand in an incubator for 40 minutes to form a fibrin gel.
  • HCM containing Endothelial Cell Growth Supplement
  • Dosing for liver model Dosing was administered 1 day (Day 1) and 4 days (Day 4) after the start of culture of the liver model by exchanging the medium with a medium containing monocotallin at a concentration of 2000 ⁇ M, 666 ⁇ M, 222 ⁇ M, or 74 ⁇ M for the liver model. Carried out. The compound used was previously dissolved in DMSO to a high concentration and stored, and DMSO was contained in the medium at a concentration of 1% at the time of dosing. Since 1% DMSO was contained in each dosing condition, medium exchange with a medium containing only 1% DMSO was also carried out at the same time as a negative control.
  • the medium containing the compound was removed from each well of 48 plates, 100 ⁇ L of DMEM at room temperature was added, and then 100 ⁇ L of ATP assay reagent attached to the kit was added at room temperature. Forty-eight plates to which ATP assay reagent was added were shaken with a constant temperature shaker for 5 minutes (1000 rpm, normal temperature). After that, 48 plates were allowed to stand at room temperature for 25 minutes.
  • BSA solution (Transparency processing / blocking) 0.2 (v / v)% TRITON / 1 (w / v)% BSA PBS solution (hereinafter referred to as BSA solution) was added to 100 ⁇ L into the insert of each well, and the mixture was allowed to stand at room temperature for 2 hours.
  • the mouse-derived anti-CD31 antibody was diluted 100-fold with BSA solution to obtain a primary antibody solution. 100 ⁇ L of the primary antibody solution was added into the insert of each well and allowed to stand at 4 ° C. for 24 hours. Then, the primary antibody solution was thoroughly washed and removed.
  • the secondary antibody was diluted 200-fold with BSA solution to obtain a secondary antibody solution. 100 ⁇ L of the secondary antibody solution was added into the insert of each well, and the mixture was allowed to stand at room temperature for 1 hour in the dark. Then, the secondary antibody solution was thoroughly washed and removed, and 100 ⁇ L of PBS was added to each well.
  • the intensities of the images taken by the above method were added up to obtain fluorescence observation images of each liver model.
  • Image analysis was performed on each of the captured images using Image J according to the following procedures (1) to (10). As a result, a skeleton image extracted from the original image was obtained. The region of the hepatic sinusoid network (blood vessel) in the skeletal image was extracted by the image cross product.
  • FIG. 13 shows the results of CD31 immunostaining 7 days after the start of culturing (Day 7) of the liver model to which the predetermined concentration of monochrome tallinn was administered.
  • FIG. 14 is a photograph of a substantially central portion 1 m 2 of the liver model extracted. As shown in FIGS. 13 to 14, it was observed that the liver sinusoid network became fragmented as the monochrome tallinn concentration was increased.
  • FIG. 15 shows the quantitative results of the total vessel length of the liver sinusoid network.
  • FIG. 16 shows the results of the ATP assay. As shown in FIGS. 15 to 16, when the hepatic sinusoid network was quantified (when the amount of damage to the vascular network was quantified), the drug efficacy could be evaluated with higher sensitivity than the ATP assay.
  • Example 8 Toxicity evaluation experiment of 18 compounds> A cell structure having a liver sinusoid network prepared using CMF (Example A) and a cell structure having a liver sinusoid network prepared using a heparin and collagen solution (Example B) were used to prepare a compound. Toxicity assessment was performed. Toxicity evaluation was performed on the compound to be evaluated by administering the compound and performing an ATP assay in the same procedure as in Test Example 6.
  • Example A The cell structure of Example A was obtained by the method described in Test Example 6.
  • Example B The cell structure of Example B was obtained by the method described in Test Example 7.
  • MOSvalue is an index used for toxicity evaluation as a value considering the maximum blood concentration (Cmax).
  • Reference example A and reference example B are reference values based on Procedure WR et al., Arch Toxicol, 2017, 91, 2849-2863 and Supplementary information of the relevant document.
  • Reference example A is 2D PHH (Two-dimensional primary human hepatocytes) in the above document
  • reference example B is 3D hLiMT (3D human liver microtissues) in the above document.
  • the IC50s of Reference Examples A and B are also shown in Tables 4-5. .. It was shown that the cell structure produced by this test example can be evaluated with good reproducibility for those reported to be toxic.
  • LKBT drug-induced liver injury

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EP4095237A4 (en) * 2020-01-20 2023-07-19 Toppan Inc. METHOD FOR PRODUCING A THREE-DIMENSIONAL CELLULAR STRUCTURE
JPWO2022114128A1 (lista_geral_produtos_quimicos.pdf) * 2020-11-26 2022-06-02
WO2022114128A1 (ja) * 2020-11-26 2022-06-02 凸版印刷株式会社 立体的細胞組織の製造方法及び立体的細胞組織
JP7239068B2 (ja) 2020-11-26 2023-03-14 凸版印刷株式会社 立体的細胞組織の製造方法及び立体的細胞組織
WO2023017800A1 (ja) * 2021-08-11 2023-02-16 凸版印刷株式会社 立体的細胞組織の製造方法及び立体的細胞組織
WO2023120573A1 (ja) * 2021-12-22 2023-06-29 凸版印刷株式会社 立体的細胞組織の培養方法

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